1. Field of the Invention
The present invention relates generally to the fields of molecular medicine and drug delivery and, more specifically, to molecules that home to a specific organ or tissue.
2. Background Information
Although the effect of a particular pathology often is manifest throughout the body of the afflicted person, generally, the underlying pathology may affect only a single organ or tissue. It is rare, however, that a drug or other treatment will target only the diseased organ or tissue. More commonly, treatment results in undesirable side effects due, for example, to generalized toxic effects throughout the patient""s body. It would be desirable to selectively target organs or tissues, for example, for treatment of diseases associated with the target organ or tissue. In particular, targeting of an organ or tissue can be useful for directing the expression of a gene to a certain organ or tissue because incorporation of a foreign gene into nontargeted cells can cause unwanted side effects such as malignant transformation.
Most therapeutic substances are delivered to the target organ or tissue through the circulation. The endothelium, which lines the internal surfaces of blood vessels, is the first cell type encountered by a circulating therapeutic substance in the target organ or tissue. These cells provide a target for selectively directing therapies to an organ or tissue.
Endothelium can have distinct morphologies and biochemical markers in different tissues. The blood vessels of the lymphatic system, for example, express various adhesion proteins that serve to guide lymphocyte homing. For example, endothelial cells present in lymph nodes express a cell surface marker that is a ligand for L-selectin and endothelial cells in Peyer""s patch venules express a ligand for the xcex14xcex27 integrin. These ligands are involved in specific lymphocyte homing to their respective lymphoid organs. Thus, linking a drug to L-selectin or to the xcex14xcex27 integrin may provide a means for targeting the drug to diseased lymph nodes or Peyer""s patches, respectively, provided that these molecules do not bind to similar ligands present in a significant number of other organs or tissues.
Although the homing molecules present in the blood vessels of non-lymphoid tissues have not been clearly defined, certain observations of lymphocyte circulation suggest that organ and tissue specific endothelial markers exist. Similarly, the homing or metastasis of particular types of tumor cells to specific organs or tissues further suggests that organ and tissue specific markers may exist. Thus, a need exists to identify molecules that can bind to such organ or tissue specific markers and, therefore, can home to the organ or tissue. The present invention satisfies this need and provides related advantages as well.
The present invention provides a method of identifying a membrane dipeptidase (MDP)-binding homing molecule, which is a molecule that selectively homes to lung endothelium. The method includes the steps of contacting MDP with one or more molecules; and determining specific binding of a molecule to the MDP, where the presence of specific binding identifies the molecule as a MDP-binding homing molecule that selectively homes to lung endothelium. In a method of the invention, the MDP can be, for example, in its natural state or substantially purified and, if desired, immobilized on a support. The membrane dipeptidase can be any mammalian MDP, for example, human MDP having SEQ ID NO: 448.
Further provided by the invention is a method of selectively directing a moiety to lung endothelium in a subject by administering to the subject a conjugate containing a moiety linked to a MDP-binding homing molecule that selectively homes to lung endothelium, whereby the moiety is selectively directed to lung endothelium in the subject. Such a method can be useful, for example, for drug targeting to lung. In a method of the invention, the MDP-binding homing molecule is identified by contacting membrane dipeptidase (MDP) with one or more molecules; and determining specific binding of a molecule to the MDP, where the presence of specific binding identifies the molecule as a MDP-binding homing molecule that selectively homes to lung endothelium. A variety of moieties can be selectively directed to lung endothelium according to a method of the invention. A moiety useful in the invention can be, for example, a gene therapy vector or drug.
In one embodiment, the invention provides a method for selectively directing a moiety to lung endothelium where the MDP-binding homing molecule is a peptide including the sequence X1-G-F-E-X2 (SEQ ID NO: 17), where X1 and X2 each is 1 to 10 independently selected amino acids. Such a MDP-binding homing peptide can include, for example, the sequence CGFECVRQCPERC (SEQ ID NO: 1) or CGFELETC (SEQ ID NO: 2).
In another embodiment, the invention provides a method for selectively directing a moiety to lung endothelium where the MDP-binding homing molecule contains the following Structure 1: 
where R2 and R3 are hydrocarbon radicals in the range respectively of 3-10 and 1-15 carbon atoms; in either one of these R2 or R3 hydrocarbon chains 1-6 hydrogens may be replaced by halogens or a nonterminal methylene may be replaced by oxygen or sulfur, including oxidized forms of the latter; additionally, a terminal hydrogen in R3 can also be replaced by hydroxyl or thiol, which may be acylated or carbamoylated; or the hydrogen can be replaced by amino, which may be derivatized as in an acylamino, ureido, amidino, quanidino, or alkyl or substituted amino group, including quaternary nitrogen grouping; or, there may be replacement by acid groups such as carboxylic, phosphonic or sulfonic acid groups or esters or amides thereof, or cyano; or combinations thereof, such as a terminal amino acid grouping; and R1 is hydrogen or lower alkyl (C1-6) or dialkylaminoalkyl, or a pharmaceutically acceptable cation. Such an MDP-binding homing molecule for reducing or preventing lung metastasis can be, for example, 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid, also known as cilastatin.
An MDP-binding homing molecule can be, for example, a compound having Structure 1 in which R2 is branched alkyl or cycloalkyl with a limitation that the carbon adjacent to the carbonyl cannot be tertiary.
An MDP-binding homing molecule also can be, for example, a compound having Structure 1 in which R2 is branched alkyl or cycloalkyl with a limitation that the carbon adjacent to the carbonyl cannot be tertiary, and in which R3 is n-alkyl (1-9 carbons) or n-alkyl (1-9 carbons) having a terminal substituent which is a quaternary nitrogen, amine derivative or amino acid derived group. An MDP-binding homing molecule can be, for example, a compound having Structure 1 in which R2 is 2,2-dimethylcyclopropyl or 2,2-dichlorocyclopropyl and in which R3 is a hydrocarbon chain of 3 to 7 carbon atoms without a terminal substituent or having a terminal substituent which is trimethylammonium, amidino, guanidino or 2-amino-2-carboethylthio.
Exemplary MDP-binding homing molecules having Structure 1 useful in the invention include the following: Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-trimethylammonium hydroxide-2-octenoic acid inner salt; Z-2-(2,2-dichlorocyclopropanecarboxamido)-8-trimethylammonium hydroxide-2-octenoic acid inner salt; Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-guanidino-2-octenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-guanidino-2-octenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-ureido-2-octenoic acid; Z-8-(1-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid (racemic and dextrorotatory forms); Z-2-(2,2-dichlorocyclopropanecarboxamido)-2-octenoic acid; 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid; and 6-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoic acid.
The present invention also provides a method of reducing or preventing lung metastasis in a subject having cancer by administering to the subject a membrane dipeptidase (MDP)-binding homing molecule. In a preferred embodiment, an MDP-binding homing molecule is a lung homing peptide including the sequence X1-G-F-E-X2 (SEQ ID NO: 17), where X1 and X2 each is 1 to 10 independently selected amino acids, such as a peptide including the sequence CGFECVRQCPERC (SEQ ID NO: 1) or CGFELETC (SEQ ID NO: 2).
In another preferred embodiment, an MDP-binding homing molecule is a molecule containing Structure 1, described hereinabove. Such an MDP-binding homing molecule can be, for example, 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid, commonly known as cilastatin.
In one embodiment, an MDP-binding homing molecule useful in the invention is an MDP inhibitor. Such an MDP inhibitor can exhibit, for example, a Ki against MDP of 1000 nM or less. In other embodiments, an MDP inhibitor useful in reducing or preventing lung metastasis exhibits a Ki against MDP of 100 nM or less or a Ki against MDP of 1 nM or less.
The present invention also provides a method of reducing or preventing lung metastasis in a subject having cancer by administering to the subject a MDP negative regulatory agent. A MDP negative regulatory agent useful in the invention can be, for example, a soluble MDP polypeptide or an antibody that selectively reacts with MDP.
Further provided herein are methods of reducing or preventing cell homing to lung endothelium in a subject by administering to the subject a MDP negative regulatory agent. A MDP negative regulatory agent useful for reducing or preventing cell homing to lung endothelium can be, for example, a soluble MDP polypeptide or an antibody that selectively reacts with MDP.
The present invention also provides a method of identifying a molecule that reduces or prevents lung metastasis by contacting membrane dipeptidase (MDP) with one or more molecules; and determining MDP activity in the presence of the molecule as compared to a control value, where diminished MDP activity in the presence of the molecule identifies the molecule as a molecule that reduces or prevents lung metastasis. The membrane dipeptidase can be, for example, substantially purified. MDP activity can be determined, for example, by release of D-Phe from Gly-D-Phe.
In one embodiment, the invention provides a method of identifying a molecule that reduces or prevents lung metastasis by contacting MDP with one or more molecules; determining MDP activity in the presence of the molecule as compared to a control value; administering the molecule to a subject having cancer; and assaying lung metastasis in the subject as compared to a control level of metastasis, where diminished MDP activity in the presence of the molecule identifies the molecule as a molecule that reduces or prevents lung metastasis.